Reagents And Methods for Detecting Protein Lysine 3-Hydroxybutyrylation

ABSTRACT

The invention provides an isolated peptide comprising a lysine 3-hydroxybutyrylation site, a lysine 3-hydroxybutyrylation specific affinity reagent that specifically binds to the peptide, and a method for detecting protein lysine 3-hydroxybutyrylation in a sample using the reagent.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to and claims the benefit of U.S. Provisional Application No. 61/866,725, entitled “REAGENTS AND METHODS FOR DETECTING PROTEIN LYSINE 3-HYDROXYBUTYRYLATION” filed 16 Aug. 2013, the contents of which are incorporated herein by reference in their entireties for all purposes.

REFERENCE TO U.S. GOVERNMENT SUPPORT

This work is supported by a grant from National Institutes of Health under Award Numbers CA126832. The United States has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to reagents and methods for detecting proteins having post-translational modifications. More particularly, it relates to peptides comprising a 3-hydroxybutyrylated lysine, and their uses to develop reagents and methods useful for detecting protein lysine 3-hydroxybutyrylation.

BACKGROUND OF THE INVENTION

Emerging lines of evidence suggest that cellular metabolism is associated with chromatin structure and epigenetic programming. Enzymes regulating histone protein post-translational modifications (PTMs), or histone marks, use high energy co-substrates, such as acetyl-CoA and S-adenosylmethionine, for protein PTM reactions. In response to the extracellular environment, the intracellular concentrations of these cofactors may change, in turn affecting the status of histone marks. In addition, the activity of histone PTM enzymes can be modulated by cellular metabolites, such as NAD and 2-hydroxyglutarate.

Histone PTMs, such as lysine acetylation, are also abundantly present in other proteins, and have diverse DNA-independent functions, including effects on metabolism¹¹. The recent discovery of new histone marks and the high complexity of cellular metabolisms imply the possibility of undescribed histone marks and PTM pathways which are modulated by metabolic signals.

3-Hydroxybutyrate is a component of ketone bodies and an important energy source for tissues during starvation. It regulates gene expression and exhibits neuroprotective effects in diverse chronic neurological diseases. However, the molecular mechanisms underlying these effects remain unclear.

There remains a need for developing reagents and methods useful for detecting post-translational modifications of histones or nonhistone proteins linked to various diseases and disorders.

SUMMARY OF THE INVENTION

The present invention relates to the use of peptides comprising a 3-hydroxybutyrylated lysine (K_(3ohbu)) to develop reagents and methods for detecting protein lysine 3-hydroxybutyrylation, especially site specific lysine 3-hydroxybutyrylation.

An isolated peptide comprising a 3-hydroxybutyrylated lysine is provided. The isolated peptide may be derived from a histone protein or a fragment thereof. The histone protein may be derived from an organism selected from the group consisting of human, mouse, S. cerevisiae, Tetrahymena thermophila, D. melanogaster, and C. elegans. The isolated peptide may comprise an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-102. The isolated peptide may comprise an amino acid sequence selected from SEQ ID NOs: 29-102. The isolated peptide may comprise at least 2 amino acid residues on each of the N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine.

An isolated lysine 3-hydroxybutyrylation specific affinity reagent is also provided. It is capable of binding specifically to a peptide comprising a 3-hydroxybutyrylated lysine. The peptide may comprise an amino acid sequence selected from SEQ ID NOs: 29-102. The binding may be dependent on the presence of the 3-hydroxybutyrylated lysine but not a surrounding peptide sequence thereof in the peptide. The binding may be dependent on the presence of the 3-hydroxybutyrylated lysine and a surrounding peptide sequence thereof in the peptide. The lysine 3-hydroxybutyrylation specific affinity reagent may be a protein or an antibody.

A method for producing a lysine 3-hydroxybutyrylation specific affinity reagent that is a protein is provided. The method comprises screening a protein library using a peptide comprising a 3-hydroxybutyrylated lysine and at least two amino acid residues on each of the N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine. The protein library may be selected from the group consisting of a phage display library, a yeast display library, a bacterial display library, and a ribosome display library.

A method for producing a lysine 3-hydroxybutyrylation specific affinity reagent that is an antibody is also provided. The method comprises immunizing a host with a peptide comprising a 3-hydroxybutyrylated lysine and at least two amino acid residues on each of the N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine.

A method for detecting a 3-hydroxybutyrylated lysine in a protein or a fragment thereof is provided. The method comprises contacting the protein or a fragment thereof with the isolated lysine 3-hydroxybutyrylation specific affinity reagent capable of binding specifically to a peptide comprising a 3-hydroxybutyrylated lysine. The lysine 3-hydroxybutyrylation specific affinity reagent and the protein or a fragment thereof forms a binding complex. The method further comprises detecting the binding complex. The presence of the binding complex indicates the presence of a 3-hydroxybutyrylated lysine in the protein or a fragment thereof. In this method, the lysine 3-hydroxybutyrylation specific affinity reagent may be a protein or an antibody.

A method for determining the level of protein lysine 3-hydroxybutyrylation in a sample is provided. The method comprises detecting a 3-hydroxybutyrylated lysine in the sample.

A kit for detecting a 3-hydroxybutyrylated lysine in a protein of a fragment thereof is provided. The kit comprises an isolated lysine 3-hydroxybutyrylation specific affinity reagent capable of binding specifically to a peptide comprising a 3-hydroxybutyrylated lysine.

A kit for isolating a peptide containing a 3-hydroxybutyrylated lysine is also provided. The kit comprises an isolated lysine 3-hydroxybutyrylation specific affinity reagent capable of binding specifically to a peptide comprising a 3-hydroxybutyrylated lysine.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 shows chemical structures of 3-hydroxybutyryllysine isomers and biosynthetic pathways. (a) Each isomer causes a predicted mass shift of +86.0368 Da. (b) Biosynthetic pathways for 3-hydroxybutyrate and 3-hydroxybutyryl-CoA. Also listed are the three ketone bodies: 3-hydroxybutyrate, acetoacetate, and acetone.

FIG. 2 shows identification and confirmation of a Lys 3-hydroxybutyrylated peptide. (a) MS/MS spectrum of a tryptic peptide derived from HEK293 core histones, KQLATK_(ac)AAR (SEQ ID NO: 29), where K_(ac) indicates acetyllysine. The peptide has a mass shift of +86.0276 Da at its Lys 1 residue. (b) MS/MS spectrum of the synthetic peptide K_(3ohbu)QLATK_(ac)AAR, where K_(3ohbu) indicates 3-hydroxybutyryllysine. (c) MS/MS spectrum of K_(3ohbu(heavy))QLATK_(ac)AAR identified from (R/S)-3-hydroxybutyrate-[2,4-¹³C2] treated HEK293 cells. The insets show the mass-to-charge ratios (m/z) of the doubly charged precursor peptide ions. (d) Reconstructed ion chromatograms from HPLC/MS/MS analyses of the in vivo-derived K_(+86.0276)QLATK_(ac)AAR peptide, its synthetic K_(3ohbu) counterpart, and their mixture, showing co-elution of the two peptides.

FIG. 3 shows detection of Lys 3-hydroxybutyrylation in cells. Western blot analysis, using a pan anti-K_(3ohbu) antibody, of (a) whole cell lysates from E. coli, S. cerevisiae, D. melanogaster S2 cells, MEF cells, and HEK293 cells, (b) liver whole-cell lysates from either control or starved (48 hours) male mice, and (c) liver whole-cell lysates from either control or STZ-treated female mice. Uniformity of sample loading was checked by staining the membrane with Ponceau S after protein transfer but prior to incubation with antibody. Concentrations of blood glucose and 3-hydroxybutyrate in (d) starved and (e) STZ-treated mice relative to controls. **P<0.01, ***P<0.001, Error bars show SD.

FIG. 4 shows proteomic screening of K_(3ohbu) substrates. (a) K_(3ohbu) sites identified on histones from HEK293 cells (red diamonds) and mouse livers (green diamonds) were mapped to the selected mouse histone sequences (UniProtKB accession number: P43277, P27661, P10853, P84244 and P62806). The modified Lys residues are highlighted in red, and sites known to be lysine-acetylated in human and mouse proteins are marked with blue squares. Cellular compartment analysis of the K_(3ohbu) proteome, showing the enrichment (b) and subcellular distribution (c) of the K_(3ohbu) substrates. (d) Analysis of the sequences surrounding 3-hydroxybutyryllysine in the K_(3ohbu) substrates from the HEK293 dataset identified SK, KxxP, KxA and KxG motifs (Bonferroni-corrected p<0.05).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of a new type of histone marks, lysine 3-hydroxybutyrylation. In particular, lysine 3-hydroxybutyrylation (K_(3ohbu)) have been identified and verified as a new, evolutionarily conserved protein post-translational modification (PTM). 3-Hydroxybutyrate can label and stimulate K_(3ohbu), presumably via conversion of 3-hydroxybutyrate to 3-hydroxybutyryl-CoA. K_(3ohbu) is a pervasive and dynamic PTM that is influenced by physiological conditions and cell status. For example, 45 non-redundant K_(3ohbu) sites in histones of HEK293 and mouse liver cells, and 3008 K_(3ohbu) sites in HEK293 cells have been identified. The present invention provide evidence to link ketone metabolism to chromatin structure, and opens up a new avenue to study the pharmacological functions and diverse roles of 3-hydroxybutyrate in pathophysiological processes.

The term “peptide” used herein refers to a linear chain of two or more amino acids linked by peptide bonds. A peptide may have about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 100, 200 or more amino acids. The amino acids of a peptide may be modified, deleted, added or substituted. A peptide may be obtained using conventional techniques known in the art. For example, a peptide may be synthesized or obtained from a native or recombinant protein by enzymatic digestion.

The term “polypeptide” used herein refers to a peptide having at least 4 amino acids, preferably at least about 20 amino acids, regardless of post-translational modification. The term “protein” used herein refers to a biological molecule consisting of one or more polypeptides, regardless of post-translational modification. Each polypeptide in a protein may be a subunit. The polypeptide or protein may be in a native or modified form, and may exhibit a biological function or characteristics.

Where a protein is a single polypeptide, the terms “protein” and “polypeptide” are used herein interchangeably. A fragment of a polypeptide or protein refers to a portion of the polypeptide or protein having an amino acid sequence that is the same as a part, but not all, of the amino acid sequence of the polypeptide or protein. Preferably, a fragment of a polypeptide or protein exhibits a biological function or characteristics identical or similar to that of the polypeptide or protein.

The term “derived from” used herein refers to the origin or source from which a biological molecule is obtained, and may include naturally occurring, recombinant, unpurified or purified molecules. A biological molecule such as a peptide (e.g., a polypeptide or protein) may be derived from an original molecule, becoming identical to the original molecule or a variant of the original molecule. For example, a peptide derived from an original peptide may have an amino acid sequence identical or similar to the amino acid sequence of its original peptide, with at least one amino acid modified, deleted, inserted, or substituted. A derived peptide may have an amino acid sequence at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, preferably at least about 50%, more preferably at least about 80%, most preferably at least about 90%, identical to the amino acid sequence of its original peptide, regardless of post-translational modification. Preferably, a derived biological molecule (e.g., a peptide) may exhibit a biological function or characteristics identical or similar to that of the original biological molecule.

The term “antibody” used herein includes whole antibodies, and antigen binding fragments (or antigen-binding portions) and single chains thereof. A whole antibody can be either one of the two types. The first type refers to a glycoprotein typically having two heavy chains and two light chains, and includes an antigen binding portion. For example, the antibody may be a polyclonal or monoclonal antibody. The term “antigen binding portion” of an antibody used herein refers to one or more fragments of the antibody that retain the ability of specifically binding to an antigen. The second type refers to a heavy-chain antibody occurring in camelids that is also called Nanobody. The term “single-chain variable fragment” of an antibody used herein refers to a fusion protein of the variable regions of the heavy and light chains of the antibody, connected with a short linker peptide, for example, of about 20-25 amino acids, that retains the ability of specifically binding to an antigen.

An isolated peptide comprising a 3-hydroxybutyrylated lysine is provided. The term “3-hydroxybutyrylated lysine” used herein refers to a lysine residue that is modified by a 3-hydroxybutyryl group at its epsilon-amine group. It may be in R-form or S-form, preferably R-form. The term “lysine 3-hydroxybutyrylation site” used herein refers to a lysine residue in a peptide, polypeptide or protein that may be 3-hydroxybutyrylated on the epsilon-amine group of the lysine residue. The term “lysine 3-hydroxybutyrylation” used herein refers to 3-hydroxysobutyrylation on the epsilon-amine group of a lysine residue that generates a 3-hydroxysobutyryl lysine residue or 3-hydroxybutyrylated lysine.

The peptide of the present invention may have at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 or 200 amino acids. The peptide may have about 3-25 amino acids, preferably 5-20 amino acids, more preferably 6-14 amino acids.

The peptide of the present invention may be prepared using conventional techniques known in the art. The peptide may be derived from a protein, for example, a histone protein, or a fragment thereof, having a lysine 3-hydroxybutyrylation site. The histone protein may be derived from a eukaryotic cell. Examples of a eukaryotic cell include cells from a yeast (e.g., S. cerevisiae), an C. elegans, a Drosophila (e.g., D. melanogaster (S2)), a Tetrahymena (e.g., Tetrahymena thermophila), a mouse (e.g., M. musculus (MEF)), or a human. Preferably, the eukaryotic cell is a mammalian cell, for example, a human, primate, mouse, rat, horse, cow, pig, sheep, goat, chicken, dog or cat cell. More preferably, the eukaryotic cell is a human cell.

The histone protein may be a histone linker protein or a histone core protein. A histone linker protein may be selected from the members of the H1 family, including the H1F subfamily (e.g., H1F0, H1FNT, H1FOO, and H1FX) and the H1H1 subfamily (e.g., HIST1H1A, HIST1H1B, HIST1H1C, HIST1H1D, HIST1H1E and HIST1H1T). A histone core protein may a member of the H2A, H2B, H3 or H4 family. A histone core protein in the H2A family may be a member of the H2AF subfamily (e.g., H2AFB1, H2AFB2, H2AFB3, H2AFJ, H2AFV, H2AFX, H2AFY, H2AFY2, and H2AFZ), the H2A1 subfamily (e.g., HIST1H2AA, HIST1H2AB, HIST1H2AC, HIST1H2AD, HIST1H2AE, HIST1H2AG, HIST1H2AH, HIST1H2AI, HIST1H2AJ, HIST1H2AK, HIST1H2AL, and HIST1H2AM), or the H2A2 subfamily (e.g., HIST2H2AA3, HIST2H2AA4, HIST2H2AB, and HIST2H2AC). A histone core protein in the H2B family may be a member of the H2BF subfamily (e.g., H2BFM and H2BFWT), the H2B1 subfamily (e.g., HIST1H2BA, HIST1H2BB, HIST1H2BC, HIST1H2BD, HIST1H2BE, HIST1H2BF, HIST1H2BG, HIST1H2BH, HIST1H2BI, HIST1H2BJ, HIST1H2BK, HIST1H2BL, HIST1H2BM, HIST1H2BN, and HIST1H2BO), or the H2B2 subfamily (e.g., HIST2H2BE and HIST2H2BF). A histone core protein in the H3 family may be a member of the H3A1 subfamily (e.g., HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, and HIST1H3J), the H3A2 subfamily (e.g., HIST2H3A, HIST2H3C, and HIST2H3D), or the H3A3 subfamily (e.g., HIST3H3), the H3A3 subfamily (e.g., H3F3A, H3F3B, and H3F3C). A histone core protein in the H4 family may be a member of the H41 subfamily (e.g., HIST1H4A, HIST1H4B, HIST1H4C, HIST1H4D, HIST1H4E, HIST1H4F, HIST1H4G, HIST1H4H, HIST1H4I, HIST1H4J, HIST1H4K, and HIST1H4L), or the H44 subfamily (e.g., HIST4H4).

The protein and gene sequences of histone proteins in various species are known in the art. For example, histone protein sequences of human, mouse, S. cerevisiae, Tetrahymena, D. melanogaster, and C. elegans can be found in GenBank database Accession Nos. GenBank database Accession No. P16403 (H1.2_HUMAN) (SEQ ID NO: 1), P0C0S8 (H2A.1_HUMAN) (SEQ ID NO: 2), P33778 (H2B.1B_HUMAN) (SEQ ID NO: 3), P84243 (H33_HUMAN) (SEQ ID NO: 4), and P62805 (H4_HUMAN) (SEQ ID NO: 5); P15864 (H12_MOUSE) (SEQ ID NO: 6), P22752 (H2A1_MOUSE) (SEQ ID NO: 7), P10853 (H2B1F/G/L_MOUSE) (SEQ ID NO: 8), P84244 (H33_MOUSE) (SEQ ID NO: 9), and P62806 (H4_MOUSE) (SEQ ID NO: 10); P04911 (H2A.1_(—) S. cerevisiae) (SEQ ID NO: 11), P02294 (H2B.2_(—) S. cerevisiae) (SEQ ID NO: 12), P61830 (H3_(—) S. cerevisiae) (SEQ ID NO: 13), and P02309 (H4_(—) S. cerevisiae) (SEQ ID NO: 14); P35065 (H2A.1_(—) Tetrahymena thermophila) (SEQ ID NO: 15), P08993 (H2B.1_(—) Tetrahymena thermophila) (SEQ ID NO: 16), I7LUZ3 (H3_(—) Tetrahymena thermophila) (SEQ ID NO: 17), and P69152 (H4_(—) Tetrahymena thermophila) (SEQ ID NO: 18); P02255 (H1_(—) D. melanogaster) (SEQ ID NO: 19), P08985 (H2A.V_(—) D. melanogaster) (SEQ ID NO: 20), P02283 (H2B_(—) D. melanogaster) (SEQ ID NO: 21), P02299 (H3) (SEQ ID NO: 22), and P84040 (H4_(—) D. melanogaster) (SEQ ID NO: 23); P10771 (H1.1_(—) c. elegans) (SEQ ID NO: 24), P09855 (H2A_(—) c. elegans) (SEQ ID NO: 25), P04255 (H2B.1_(—) c. elegans) (SEQ ID NO: 26), P08898 (H3_(—) c. elegans) (SEQ ID NO: 27), and P62784 (H4_(—) c. elegans) (SEQ ID NO: 28).

A fragment of a histone protein may have an amino acid sequence that is the same as a part, not all, of the amino acid sequence of the histone protein comprising at least one lysine 3-hydroxybutyrylation site. The histone protein fragment may have at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 or 200 amino acids. The histone fragment may have about 3-25 contiguous amino acids, preferably about 5-20 contiguous amino acids, more preferably about 6-14 contiguous amino acids, of the histone protein covering at least one lysine 3-hydroxybutyrylation site in the histone protein.

The histone protein or fragment may have a 3-hydroxybutyrylated lysine at a lysine 3-hydroxybutyrylation site. The lysine 3-hydroxybutyrylation site may be any one of the lysine 3-hydroxybutyrylation sites in exemplary histone proteins of human (Table 1) and mouse (Table 2).

A histone protein may be obtained from a biological sample or prepared using recombinant techniques. A histone protein fragment may be prepared by recombinant techniques, or by digesting the histone protein with an enzyme (e.g., trypsin). The lysine 3-hydroxybutyrylation site in the histone protein or fragment may be lysine 3-hydroxybutyrylated naturally or artificially. The presence of a 3-hydroxybutyrylated lysine may be confirmed by using conventional techniques known in the art, for example, mass spectrometry.

The peptide of the present invention may comprise an amino acid sequence having at least about 70%, 80%, 90%, 95% or 99%, preferably at least about 90%, more preferably 100%, identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-102. The peptide may encompass any lysine 3-hydroxybutyrylation site with or without its surrounding sequences from a histone proteins. The peptide may comprise more than one 3-hydroxybutyrylated lysine. The peptide may also comprise a protein post-translational modification other than 3-hydroxybutyrylated lysine, such as acetylated lysine or methylated lysine. The peptides may further comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues on either or both of N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine. Preferably, the peptide may comprise at least 2 amino acid residues on each of the N-terminal and C-terminal side of the 3-hydroxybutyrylated lysine. Exemplary peptides of the present invention are shown in Tables 1 and 2.

An isolated lysine 3-hydroxybutyrylation specific affinity reagent is also provided. The term “lysine 3-hydroxybutyrylation specific affinity reagent” used herein refers to a molecule that is capable of binding to a peptide, polypeptide or protein having a lysine 3-hydroxybutyrylation site, which may be a histone protein or a peptide of the present invention. The lysine 3-hydroxybutyrylation specific affinity reagent may be a protein, for example, an antibody. The lysine 3-hydroxybutyrylation site may be any lysine 3-hydroxybutyrylation site in any histone protein from any species. Examples of the lysine 3-hydroxybutyrylation sites include those in human (Table 1) and mouse (Table 2), and homologous lysine sites in corresponding eukaryotic histone proteins.

In some embodiments, the lysine 3-hydroxybutyrylation specific affinity reagent binds a peptide, polypeptide or protein having a lysine 3-hydroxybutyrylation site that is 3-hydroxybutyrylated, either in R-form or S-form, preferably in R-form, having an affinity that is at least about 10, 50, 100, 500, 1000 or 5000 times higher than that for its counterpart when the site is not 3-hydroxybutyrylated.

In other embodiments, the lysine 3-hydroxybutyrylation specific affinity reagent binds a peptide, polypeptide or protein having a lysine 3-hydroxybutyrylation site that is not 3-hydroxybutyrylated, having an affinity that is at least about 10, 50, 100, 500, 1000 or 5000 times higher than that for its counterpart when the site is 3-hydroxybutyrylated, either in R-form or S-form, preferably in R-form. The lysine 3-hydroxybutyrylation specific affinity reagent may be a peptide, polypeptide or protein, which may be an antibody. Preferably, the peptide is a peptide of the present invention.

The lysine 3-hydroxybutyrylation specific affinity reagent may be site specific, i.e., the binding is dependent on the presence of the 3-hydroxybutyrylated lysine, either in R-form or S-form, preferably in R-form, and its surrounding peptide sequence. The surrounding peptide sequence may include at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues on either or both of N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine. For example, the binding depends on the presence of the 3-hydroxybutyrylated lysine and at least 2 amino acid residues on each of the N-terminal and C-terminal side of the 3-hydroxybutyrylated lysine.

The lysine 3-hydroxybutyrylation specific affinity reagent may not be site specific, i.e., the binding is dependent on the presence of the 3-hydroxybutyrylated lysine but not its surrounding peptide sequence. One example is an anti-lysine-3-hydroxybutyrylation pan antibody.

A method for producing the lysine 3-hydroxybutyrylation specific affinity reagent of the present invention is further provided.

Where the lysine 3-hydroxybutyrylation specific affinity reagent is a protein, the protein may be produced by screening a protein library (also known as a display library or a degenerated protein library) using the peptide of the present invention. The peptide may have at least two amino acid residues one each of the N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine. The protein library may consist of many degenerated protein sequences, which may comprise two regions: one or more fixed peptide sequence regions and a plurality of degenerated amino acid sequences. The protein library may be a phage protein library, a yeast protein library, bacterial protein library, ribosome protein library, or other synthetic protein library comprising peptides having randomized amino acid sequences.

Where the lysine 3-hydroxybutyrylation specific affinity reagent is an antibody, the antibody may be produced by different methods known in the art. For example, the production method may comprise immunizing a host with an antigenic peptide to produce the antibody. The method may further comprise collecting antisera from the host. The host may be a mammal suitable for producing antibodies. For example, the host may be a mouse, rabbit, goat, Camelidae family animal (such as Lama and camel), or cartilaginous fishes. Dependent on the host used, the generated antibody can contain either two chains (a heavy chain and a light chain) or one chain (or heavy chain-only antibody occurring in camelids) that is also called Nanobody.

The antigenic peptide may be derived from a histone protein or a fragment thereof comprising a lysine 3-hydroxybutyrylation site, which may be 3-hydroxybutyrylated or not. The antigenic peptide may comprise a peptide of the present invention. Examples of antigenic peptides having 3-hydroxybutyrylated lysine may comprise one or more of the peptides in Tables 1 and 2. Examples of antigenic peptides not having 3-hydroxybutyrylated lysine may have an amino acid sequence identical to those in Tables 1 and 2, except that the lysine 3-hydroxybutyrylation site is not 3-hydroxybutyrylated. The N-terminal or C-terminal end of any of these peptides may be extended by 1-20 residues.

The method may further comprise purifying the antibody from the antisera. The method may further comprise utilizing spleen cells from the host to generate a monoclonal antibody. In some embodiments, the antibody specifically binds to a histone protein or fragment having a lysine 3-hydroxybutyrylation site when the site is 3-hydroxybutyrylated, but not when the site is not 3-hydroxybutyrylated. In other embodiments, the antibody specifically binds to a histone protein or fragment having a lysine 3-hydroxybutyrylation site when the site is not 3-hydroxybutyrylated, but not when the site is 3-hydroxybutyrylated.

The method may further comprise deduce the antibody sequences by high-performance liquid chromatography (HPLC)-mass spectrometry analysis of the isolated antibodies and followed by protein sequence database search against all the possible IgG protein sequences (derived from cDNA sequences) from bone marrow (or B cells) of the immunized host. The IgG cDNA sequences can be obtained from conventional DNA sequencing technologies from IgG cDNAs that are generated by RT-PCR using the known art. The derived heavy- and light-chain variable regions (VH and VL) can be further paired (in case the IgG is from a two-chain antibodies from a host like mice or rabbit). Such a pairing is not necessary for those IgG derived from heavy chain-only antibody (or Nonabody) from Lama. The antibody can then be generated using the antibody sequence information using the known art.

A method for detecting a 3-hydroxybutyrylated lysine in a protein or its fragment is provided. The 3-hydroxybutyrylated lysine may be R-3-hydroxybutyrylated lysine or S-3-hydroxybutyrylated lysine, preferably R-3-hydroxybutyrylated lysine. The method comprises (a) contacting the protein or its fragment with a lysine 3-hydroxybutyrylation specific affinity reagent of the present invention to form a binding complex, and (b) detecting the binding complex. The presence of the binding complex indicates the presence of the 3-hydroxybutyrylated lysine in the protein or its fragment. The binding complex may be detected by using various conventional methods in the art. The protein may be a histone protein. The method may further comprise quantifying the amount of the binding complex. The amount of the binding complex may indicate the level of lysine 3-hydroxybutyrylation in the protein or its fragment.

For each detection method of the present invention, a kit is provided. The kit comprises a lysine 3-hydroxybutyrylation specific affinity reagent of the present invention. The lysine 3-hydroxybutyrylation specific affinity reagent may be R-lysine 3-hydroxybutyrylation specific affinity reagent or S-lysine 3-hydroxybutyrylation specific affinity reagent. The kit may further comprise an instruction directing how to carry out the method.

A fusion protein reporter is provided. The fusion protein reporter comprises a core flanked by a donor fluorescent moiety and an acceptor fluorescent moiety. The core includes a peptide, which comprises a lysine 3-hydroxybutyrylation site and a lysine 3-hydroxybutyrylation binding domain. The term “lysine 3-hydroxybutyrylation binding domain” used herein refers to a region in a protein sequence capable of specific binding to the lysine 3-hydroxybutyrylation site.

The fusion protein reporter of the present invention may be useful for determining protein lysine 3-hydroxybutyrylation level in a sample or screening for an agent that regulates protein lysine 3-hydroxybutyrylation by using the fluorescence resonance energy transfer (FRET). The FRET involves the transfer of photonic energy between fluorophores when in close proximity. Donor fluorescent moieties and acceptor fluorescent moieties suitable for FRET are known in the art. In the fusion protein reporter, the donor fluorescent moiety may be selected from the group consisting of cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), and A206K mutants thereof, and the acceptor fluorescent moiety may be selected from the group consisting of yellow fluorescent protein (YFP), enhanced yellow fluorescence protein (EYFP), Citrine, Venus, and A206K mutants thereof.

The peptide in the fusion protein reporter may comprise a peptide of the present invention. It may be derived from a histone protein or fragment comprising a lysine 3-hydroxybutyrylation site, where the histone protein or fragment may be 3-hydroxybutyrylated or not at the lysine 3-hydroxybutyrylation site.

The lysine 3-hydroxybutyrylation site may be located in the N-terminus, C-terminus or the core region of a histone protein. The N-terminus, C-terminus, and core regions of histone proteins (e.g., human or mouse H1.2, H2A, H2B, H3 or H4) are known in the art.

The fusion protein reporter may comprise one or more lysine 3-hydroxybutyrylation binding domains. A lysine 3-hydroxybutyrylation binding domain may be derived from a lysine 3-hydroxybutyrylation specific affinity reagent of the present invention.

In some embodiments, the lysine 3-hydroxybutyrylation site in the peptide is not 3-hydroxybutyrylated, and the lysine 3-hydroxybutyrylation binding domain specifically binds to the lysine 3-hydroxybutyrylation site when the site is 3-hydroxybutyrylated, but not when the sites is not 3-hydroxybutyrylated.

In other embodiments, the lysine 3-hydroxybutyrylation site in the peptide is 3-hydroxybutyrylated, and the lysine 3-hydroxybutyrylation binding domain specifically binds to the lysine 3-hydroxybutyrylation site when the peptide is not lysine 3-hydroxybutyrylated, but not when the site is 3-hydroxybutyrylated.

The lysine 3-hydroxybutyrylation site may be conjugated to the lysine 3-hydroxybutyrylation binding domain with a linker molecule. The linker molecule may be a peptide have any amino acid sequence, and may have about 1-50 amino acids, preferably 1-30 amino acids, more preferably 2-15. In some embodiments, the linker molecule may be -Gly-Gly-. The length and contents of a linker molecule may be adjusted to optimize potential fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor fluorescent moiety when the lysine 3-hydroxybutyrylation site in the fusion protein reporter is 3-hydroxybutyrylated or not, and bound by the lysine 3-hydroxybutyrylating binding domain.

The fusion protein reporter may further comprise a targeting polypeptide. The targeting polypeptide may be selected from the group consisting of a receptor ligand, a nuclear localization sequence (NLS), a nuclear export signal (NES), a plasma membrane targeting signal, a histone binding protein, and a nuclear protein.

A method for determining the level of protein lysine 3-hydroxybutyrylation in a sample. The method comprises detecting a 3-hydroxybutyrylated lysine in the sample. The method may comprise (a) contacting the sample with a fusion protein reporter of the present invention, and (b) comparing the level of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor fluorescent moiety after contacting with that before contacting. The level of FRET indicates the level of protein lysine 3-hydroxybutyrylation in the sample. The level of FRET may be increased or decreased after contacting.

A method for determining the level of protein de-lysine-3-hydroxybutyrylation in a sample is also provided. The method comprises (a) contacting the sample with a fusion protein reporter of the present invention, and (b) comparing the level of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor fluorescent moiety after contacting with that before contacting. The level of FRET indicates the level of protein de-lysine-3-hydroxybutyrylation in the sample. The level of FRET may be increased or decreased after contacting.

For the determination method of the present invention, a sample may be a biological sample (e.g., bodily fluid or serum). The biological sample may comprise a cell, a tissue biopsy, or a clinical fluid. The biological sample may be obtained from a subject (e.g., a mouse, rat, or human). The subject is healthy. The subject may have suffered from or may be predisposed to a protein lysine 3-hydroxybutyrylation or de-lysine-3-hydroxybutyrylation related disorder, which may be any disorder or disease linked to abnormal regulation of protein lysine 3-hydroxybutyrylation or de-lysine-3-hydroxybutyrylation, respectively. Examples of such disorder or disease may include cancer, neurodegenerative diseases, aging, metabolic disorder, and dysgenesis.

The determination method of the present invention may further comprise comparing the FRET level in the sample with a control FRET level. The control FRET level may be the FRET level in a control sample obtained from a subject, who is healthy or has not suffered from or predisposed to a protein lysine 3-hydroxybutyrylation related disorder. The FRET level in the sample may be higher or lower than the control FRET level.

The determination method of the present invention may further comprise adding an agent to the sample. In some embodiments, the agent is known to promote or inhibit protein lysine 3-hydroxybutyrylation. In other embodiments, the agent is a screening candidate for a regulator of protein lysine 3-hydroxybutyrylation. The screening candidate may be a compound or a biological molecule.

For each determination method of the present invention, a kit is provided. The kit comprises a fusion protein of the present invention. The kit may further comprise an instruction directing how to carry out the method.

A kit for isolating a peptide containing a 3-hydroxybutyrylated lysine is also provided. The kit comprises an isolated lysine 3-hydroxybutyrylation specific affinity reagent capable of binding specifically to a peptide comprising a 3-hydroxybutyrylated lysine.

A method for treating or preventing a protein lysine 3-hydroxybutyrylation related disease in a subject in need thereof is provided. The method comprises administering to the subject an effective amount of a composition comprising an agent that regulates protein lysine 3-hydroxybutyrylation. The agent may be a screen candidate identified by a determination method of the present invention. The protein lysine-3-hydroxybutyrylation may be histone lysine-3-hydroxybutyrylation.

A method for treating or preventing a protein or de-lysine-3-hydroxybutyrylation related disease in a subject in need thereof is provided. The method comprises administering to the subject an effective amount of a composition comprising an agent that regulates protein de-lysine-3-hydroxybutyrylation. The agent may be a screen candidate identified by a determination method of the present invention. The protein de-lysine-3-hydroxybutyrylation may be histone de-lysine-3-hydroxybutyrylation.

Example 1 Materials and Methods

Peptide Sample Preparation

Synthesis and characterization of modified lysine residues used for peptide synthesis is described in the Additional Methods published online. Trypsin digestion of histones and whole-cell lysate samples was performed as previously described. Ten milligrams of whole-cell lysate tryptic digest was separated into 80 fractions with basic reversed phase HPLC. The peptide fractions were concatenated into 20 fractions and subjected to immunoaffinity enrichment for K_(3ohbu) peptides using a similar method previously described²⁸.

MS/MS Data Analysis

Peptide sample was analysed by HPLC-MS/MS and the data was searched against an IPI human (v3.70) or IPI mouse (v3.74) database. Bioinformatic analyses were performed as previously described with a Benjamini-Hochberg false discovery rate of 1%. Detailed methods were described in the Additional Methods published online.

Cell Culture and Animal Experiments

HEK293 cells were grown in complete DMEM medium either not treated, or treated with chemicals at conditions specified elsewhere in the text. C57BL/6 mice were either fed with standard chow diet, or fasted (with free access to water) for a specified number of hours as detailed in the text. C57BKS/J db/db littermates (licensed by the Jackson Laboratory) were either given single-dose intraperitoneal injections of streptozotocin (STZ, 200 mg/kg body weight), or the sodium citrate buffer vehicle for 48 hours. The liver tissues were collected for histone extraction and western blot analysis.

Reagents

The pan anti-K_(3ohbu) antibody was co-developed with PTM Biolabs, Inc. (Chicago, Ill.). Peptides were synthesized using racemic or enantiomeric modifier protected amino acid residues. Synthesis of Fmoc-protected amino acid residues is described in detail in Supplementary Methods. Modified sequencing-grade trypsin was purchased from Promega (Madison, Wis.). C18 ZipTips were bought from Millipore Corporation (Bedford, Mass.). Other chemicals were obtained from the following suppliers. Sigma-Aldrich (St. Louis, Mo.): formic acid (>98%), NH₄HCO₃ (>99%), trichloroacetic acid (6.1 N), iodoacetamide, dithiothreitol, bovine serum albumin, sodium butyrate, nicotinamide, trichostatin A, Fmoc-Lys-OH (98%), Sodium (R/S)-3-Hydroxybutyrate-2,4-¹³C2 (99 atom %), (R/S)-3-hydroxybutyrate, (R)-3-hydroxybutyrate, (S)-3-hydroxybutyrate, 2-hydroxyisobutyric acid (98%), N-hydroxysuccinimide (98%), H₃PO₄ (99%), BF₃.OEt₂, isobutylene (99%), LiOH (>98%), 4-butyrolactone (>99%), trityl chloride (98%), anhydrous pyridine (99.8%), ethyl (R)-3-hydroxybutyrate (98%), ethyl (S)-3-hydroxybutyrate (99%), N,N′-dicyclohexylcarbodiimide (DCC) (99%), anhydrous dioxane (99.8%), methyl (S)-3-hydroxy-2-methylpropionate (99%), methyl (R)-3-hydroxy-2-methylpropionate (99%), trifluoroacetic acid (99%), and ethyl 2-hydroxybutyrate (>95% GC). Fisher (Pittsburgh, Pa.): NaHCO₃ (ACS grade), NaOH (ACS grade), CH₃CN (HPLC grade), CH₂Cl₂ (HPLC grade), HCl solution (37.3%), MeOH (ACS grade), acetone (ACS grade), EtOAc (ACS grade), anhydrous Et₂O (ACS grade), DMEM medium (high glucose), anhydrous Na₂SO₄ (ACS grade), anhydrous MgSO₄ (ACS grade), hexane (ACS grade), Et₃N (>99%), and hydrogen peroxide. Abcam: anti-histone H3 antibody, anti-alpha tubulin antibody.

Cell Culture and Preparation of Peptide Samples

HEK293 cells were grown to 90% confluence in complete DMEM medium at 37° C. in a humidified incubator supplemented with 5% CO₂. For isotopic labelling, HEK293 cells were grown in complete DMEM medium containing 20 mM (R/S)-3-hydroxybutyrate [2,4-¹³C2] for 48 hrs until they reached 95% confluence. For identification of 3-hydroxybutyrylation substrates, HEK293 cells were grown in complete DMEM medium treated with or without sodium 3-hydroxybutyrate as specified elsewhere in the paper. The cells were lysed in lysis buffer (100 mM NaCl, 20 mM Tris, 0.5 mM EDTA, 0.5% (v/v) NP40, 0.2 mM PMSF, 2 μg/μL leupeptin, 10 μg/mL aprotinin, 5 mM sodium butyrate and 10 mM nicotinamide) at 4° C. for 20 min with constant rotation. The sample was centrifuged for 10 min at 4° C. at 20,000×g. The insoluble pellet was resuspended in 10 volumes of lysis buffer followed by brief sonication at 4° C. The protein lysate samples were combined and precipitated in 80% cold acetone (pre-chilled to −20° C.) and 10% trichloroacetic acid solution at −20° C. for 2 hrs. The protein pellet was washed twice with cold acetone and the sample was digested with 50:1 sequencing grade modified trypsin (Promega) at 37° C. for 16 hrs. The digestion was reduced with 5 mM dithiothreitol at 50° C. for 30 min, alkylated with 15 mM iodoacetamide at rt for 30 min, and blocked with 30 mM cysteine at rt for 30 min. After reduction and alkylation, the sample was digested with 100:1 trypsin at 37° C. for an additional 4 hrs. For proteomic identification of K_(3ohbu) substrates from HEK293 cells, the tryptic peptides were further separated by reversed phase chromatography as described below.

Animal Experiments

All animal experiments were approved by the Animal Ethics Committee of the Shanghai Institute of Materia Medica, China, where the experiments were conducted. For the starvation experiment, two groups of 16 weeks old adult C57BL/6 mice (control group: n=20, 10 males and 10 females; experimental group: n=40, 20 males and 20 females) were either fed with standard chow diet containing 19% protein, or fasted (with free access to water) for 24, 48 and 72 hours (9:00 am to 9:00 am) as detailed in the text.

C57BKS/J db/db mice were licensed from the Jackson Laboratory and bred in house. All mice were housed in a temperature-controlled room (22±2° C.), with a light/dark cycle of 12 h/12 h. At the age of 12 weeks, C57BKS/J db/db littermates (C57BLKS/J lean mice) were recruited to the experiment and randomly assigned. For the ketoacidosis experiment, db-littermate mice (n=6 for each group, 3 males and 3 females) were either given single-dose intraperitoneal injections of streptozotocin (STZ, 200 mg/kg body weight), or the sodium citrate buffer vehicle. After 48 hrs (9:00 am to 9:00 am), blood samples were taken from a tail vein for determination of blood glucose and 3-hydroxybutyrate concentrations using a gluco-ketone meter (Lifescan, Burnaby, BC, Canada). The mice were decapitated and liver tissue was collected for histone extraction and lysate preparation.

Extraction of Histones

Extraction of core histones from HEK293 cells and mouse liver was carried out according to a previously described protocol with minor modifications. Liver samples were homogenized using a glass Dounce homogenizer (20 strokes) in ice-cold lysis buffer. The homogenate was passed through two layers of cheese cloth and then centrifuged at 1,000×g at 4° C. for 5 min. The pellet was briefly washed with lysis buffer and extracted with 0.4 NH₂SO₄ at 4° C. overnight. HEK293 cells were lysed in lysis buffer on ice for 10 min with gentle stirring. The lysate was removed and the pellet was washed once with the lysis buffer and then extracted with 0.4 N H₂SO₄ at 4° C. overnight. The suspension was centrifuged at 20,000×g for 10 min at 4° C. The histone-containing supernatants were precipitated with 20% trichloroacetic acid. The precipitated histone pellets were washed twice with cold acetone and dried. The histone samples were then digested with sequencing grade trypsin as described earlier.

Reversed Phase Fractionation of Tryptic Peptides

Peptide fractionation by reversed-phase chromatography was performed on a Phenomenex Luna C18 column (10 mm×250 mm, 5 μm particle, 100 Å pore size) with a flow rate of 4 mL/min using the Shimadzu preparative HPLC system. Buffer A consisted of 10 mM ammonium formate in water (pH 7.8) and buffer B consisted of 10 mM ammonium formate in 90% acetonitrile (pH 7.8). Peptides were loaded onto the column in 2 mL of buffer A and eluted with a gradient of 2-30% B in 40 min and 30-90% B in 10 min. A total of 80 fractions were collected and concatenated into 20 fractions. Acetonitrile was removed from each fraction using a Rotavapor evaporator connected to a water pump; the remaining samples were dried by lyophilisation. Immunoaffinity enrichment of K_(3ohbu) peptides was performed as previously described.

HPLC-Mass Spectrometry Analysis

Tryptic peptides were dissolved in HPLC buffer A (0.1% formic acid in water) and loaded onto a self-packed C18 capillary column (10 cm in length, 75 μm ID) packed with Jupiter C12 resin (Phenomenex, 90 Å, 4 μm in size) by Eksigent 1D-plus nano-flow HPLC. Peptides were eluted with a linear gradient of 5%-30% B in 2 hrs with a constant flow rate of 200 nL/min. Peptide ions were directly electrosprayed into a LTQ Velos Orbitrap mass spectrometer and analysed by either fragmenting the 20 most intense ions in a data-dependant mode, or fragmenting specified precursor ions for targeted analysis by collision-induced dissociation.

Peptide Identifications and Quantifications

MS/MS data were analyzed by Maxquant (v1.3.0.5) with a built-in Andromeda search engine against an IPI human (v3.70) or IPI mouse (v3.74) database for protein and peptide identification. Lys acetylation, 3-hydroxybutyrylation, methionine oxidation, and protein N-terminal acetylation were specified as variable modifications. Mass tolerance was set to 6 ppm for precursor ions and 0.5 Da for fragment ions. Results were filtered at a 1% false discovery rate at protein, peptide and site levels. To reduce the number of low quality PTM identifications, we further remove all peptides with Maxquant peptide score below 60 or site localization probability below 0.9. We also removed all peptide identification with C-terminal Lys modifications and peptide identifications from known contaminant proteins.

Bioinformatic Analysis

Statistical gene enrichment and under-enrichment analysis for Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Pfam domains was performed as previously described with a Benjamini-Hochberg false discovery rate of 1%. The manually curated CORUM protein complex database for mouse and human was used for protein complex analysis. Overrepresented complexes were identified using the hypergeometric test with a Benjamini-Hochberg false discovery rate of 1%, and were visualized in Cytoscape. Protein-protein interactions involving Lys-3-hydroxybutyrylated proteins were extracted from the STRING database and interaction sub-networks were identified by the MCODE plug-in tool in Cytoscape. Flanking sequence preference was analyzed by Icelogo with p<0.05. Sequence motif identification was performed by motif-x (Bonferroni p<0.05) and visualized by Weblogo.

Synthesis and Characterization of Modified Lysine Residues Used for Peptide Synthesis

Fmoc-Lys(2-hydroxyisobutyryl)-OH. DCC (32.6 mM, 6.72 g) was added to a solution of 2-hydroxyisobutyric acid (32 mM, 3.35 g) and N-hydroxysuccinimide (32.6 mM, 3.75 mg) in 30 mL anhydrous CH₃CN. The resulting mixture was stirred at rt for 3 hrs and then filtered. The filtration was evaporated to dryness. The residue was redissolved in 300 mL CH₂Cl₂, and Et₃N (64 mM, 8.9 mL) and Fmoc-Lys-OH (32 mM, 12.9 g) were added. The mixture was stirred at rt for 8 hrs. The solvent was evaporated and the residue was redissolved in water. The pH of the solution was adjusted to 2.0. The aqueous solution was extracted 3 times with ethyl acetate. The organic phases were combined, washed with brine and dried over anhydrous Na₂SO₄. The solvent was reduced to dryness and the residue was purified through flash column chromatography (eluent: MeOH/CH₂Cl₂=1/40 to 1/20). A yield of 8.7 g (60%) Fmoc-Lys(2-hydroxyisobutyryl)-OH was obtained. Fmoc-Lys(2-hydroxyisobutyryl)-OH: ¹H NMR (500 MHz, CDCl₃): δ 7.69 (d, J=7.5 Hz, 2H), 7.54 (t, J=7.5 Hz, 2H), 7.33 (t, J=7.5 Hz, 2H), 7.23 (t, J=7.5 Hz, 2H), 5.99 (d, J=8.0 Hz, 1H), 4.30-4.40 (m, 3H), 4.12-4.14 (m, 1H), 3.18-3.19 (m, 2H), 1.64-1.84 (m, 2H), 1.37-1.47 (m, 10H); ¹³C NMR (125 MHz, CDCl₃): δ 177.6, 174.9, 156.4, 143.7 (143.6), 141.2 (141.1), 127.7, 127.0, 125.0, 119.9, 73.5, 67.1, 53.6, 47.0, 38.6, 31.6, 28.7, 27.5 (27.4), 22.1; IR (KBr): 3361.7, 2934.8, 2868.3, 1711.2, 1696.9, 1642.5, 1631.6, 1536.4, 1454.6, 1270.5, 1254.9, 1200.1, 1176.6, 1047.3, 765.4, 740.3 cm⁻¹; HRMS (m/z): [M]⁺ calcd. for C₂₅H₃₁N₂O₆, 455.2182. found, 455.2165.

Fmoc-Lys((±)-2-(^(t)BuO) butyryl)-OH. Step 1: Racemic ethyl 2-hydroxybutyrate (15.1 mM, 1.78 g, 1.83 mL) was dissolved in 25 mL CH₂Cl₂. Then, 2 g of 99% H₃PO₄ and 312 μL BF₃.OEt₂ was added in sequential order. The resulting mixture was cooled in an ice-acetone bath and stirred. Then, 10 mL isobutylene measured in a 50 mL cylinder (pre-cooled in a dry ice acetone bath) was poured into the flask. The flask was sealed and the reaction was stirred under −78° C. for 1 hr, then allowed to return to rt. After 10 hrs, the isobutylene was discharged and the solvent was evaporated to dryness. The residue was redissolved in 100 mL EtOAc. The solution was washed 3 times with saturated NaHCO₃ solution, followed by brine. The organic layer was separated and dried over anhydrous Na₂SO₄. The solvent was evaporated and the residue was purified through flash column chromatography. The column was first eluted with 50 mL hexane/triethylamine (v/v=50/1), then with 200 mL hexane/CH₂Cl₂ (v/v=7/1), followed by 200 mL hexane/EtOAc (v/v=8/1). A yield of 1.31 g (50%) ethyl 2-(^(t)BuO) butyrate was obtained.

Step 2: Ethyl 2-(^(t)BuO) butyrate (1.31 g, 7 mM) was dissolved in 20 mL MeOH/H₂O=1/1 solution. Then, LiOH (25 mM, 575 mg) was added. The mixture was stirred at rt for 1.5 hrs. Fifty milliliters of water was added. The solution was washed with Et₂O (30 mL×2) to remove some impurities. Then, the aqueous layer was separated and acidified with 1 M HCl solution to pH 2.3. The aqueous solution was extracted 3 times with EtOAc. The organic layer was combined, washed with brine and dried over anhydrous Na₂SO₄. The solvent was filtered and evaporated to give the crude product 2-(^(t)BuO) butyric acid which was used in the next step without further purification.

Step 3: DCC (6.5 mM, 1.31 g) was added to a solution (80 mL) of 2-(^(t)BuO) butyric acid (1.04 g, 6.5 mM) and N-hydroxysuccinimide_(—) (6.5 mM, 748 mg) in CH₃CN. The reaction was stirred at rt for 4 hrs. The resulting suspension was filtered and concentrated under vacuum. The residue was redissolved in 100 mL CH₂Cl₂. Et₃N (13 mM, 1.81 mL) and Fmoc-Lys-OH (6.5 mM, 2.62 g) were sequentially added. The reaction mixture was stirred at rt for 8 hrs and then evaporated, and the residue was dissolved in 150 mL water. The solution was adjusted to pH 2.3. The organic layer was separated and the aqueous layer was extracted 3 times with EtOAc. The combined organic layers were dried over anhydrous Na₂SO₄. The residue was purified through flash column chromatography (eluent: MeOH/CH₂Cl₂=1/40 to 1/20). A yield of 2.5 g (75%) Fmoc-Lys(2-(^(t)BuO) butyryl)-OH was obtained. ¹H NMR (500 MHz, CDCl₃): δ 9.73 (s, 1H), 7.73 (d, J=7.5 Hz, 2H), 7.54-7.61 (m, 2H), 7.36 (t, J=7.5 Hz, 2H), 7.28 (t, J=7.5 Hz, 2H), 6.87-6.90 (m, 1H), 5.85 (dd, J=27.5, 9.0 Hz, 1H), 4.28-4.44 (m, 3H), 4.15-4.19 (m, 1H), 3.91-3.94 (m, 1H), 3.19-3.36 (m, 2H), 1.38-1.92 (m, 8H), 1.17 (1.15) (s, 9H), 0.88 (q, J=7.0 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃): δ 175.7, 174.6, 156.1, 143.87 (143.86, 143.70, 143.68), 141.2, 127.6, 127.0, 125.2 (125.1), 119.8, 75.3 (75.2), 73.6 (73.5), 67.0, 53.6, 47.1, 38.4 (38.4), 31.84 (31.2), 29.22 (29.16), 27.89 (27.87), 27.67 (27.66), 22.22 (22.18), 9.42 (9.36); IR (KBr): 3404, 3336, 3065, 2973, 2936, 2875, 1718, 1632, 1538, 1451, 1338, 1255, 1192, 1107, 1081, 1057, 1005, 760, 740 cm⁻¹; HRMS (m/z): [M]⁺ calcd. for C₂₉H₃₉N₂O₆, 511.2808. found, 511.2787.

Fmoc-Lys((S)-3-(^(t)BuO) isobutyryl)-OH, Fmoc-Lys((R)-3-(^(t)BuO) isobutyryl)-OH, Fmoc-Lys((R)-3-(^(t)BuO) butyryl)-OH and Fmoc-Lys((S)-3-(^(t)BuO) butyryl)-OH were synthesized in a similar manner to Fmoc-Lys((±)-2-(^(t)BuO) butyryl)-OH, starting from different raw materials. Fmoc-Lys((±)-3-(^(t)BuO) isobutyryl)-OH: ¹H NMR (500 MHz, CDCl₃): δ 9.76 (s, 1H), 7.72 (d, J=7.5 Hz, 2H), 7.58 (dd, J=10.0, 8.0 Hz, 2H), 7.35 (t, J=7.5 Hz, 2H), 7.26 (t, J=7.5 Hz, 2H), 6.85 (t, J=6.0 Hz, 1H), 5.91 (t, J=8.0 Hz, 1H), 4.29-4.41 (m, 3H), 4.16-4.18 (m, 1H), 3.38-3.39 (m, 2H), 3.18-3.30 (m, 2H), 2.42-2.48 (m, 1H), 1.47-1.94 (m, 2H), 1.41-1.53 (m, 4H), 1.14 (s, 9H), 1.11 (d, J=7.5 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃): δ 176.2 (176.1), 174.6, 156.2, 143.8 (143.7), 141.1, 127.5, 126.9, 125.09 (125.05), 119.8, 73.6, 66.9, 63.8 (63.7), 53.6, 47.0, 40.99 (40.97), 38.8, 31.7, 29.0, 27.265 (27.260), 22.2, 14p.0 (13.9); IR (KBr): 3334, 3066, 2974, 2927, 2872, 1726, 1657, 1541, 1450, 1364, 1335, 1235, 1028, 876, 760, 739 cm⁻¹; HRMS (m/z): [M]⁺ calcd. for C₂₉H₃₉N₂O₆, 511.2808. found, 511.2791.

Fmoc-Lys((S)-3-(^(t)BuO) butyryl)-OH: ¹H NMR (500 MHz, CDCl₃): δ 9.23 (s, 1H), 7.73 (d, J=7.5 Hz, 2H), 7.58 (t, J=7.5 Hz, 2H), 7.36 (t, J=7.5 Hz, 2H), 7.27 (t, J=7.5 Hz, 2H), 6.87-6.89 (m, 1H), 5.85 (d, J=8.0 Hz, 1H), 4.33-4.46 (m, 3H), 4.16-4.19 (m, 1H), 4.01-4.07 (m, 1H), 3.31-3.38 (m, 1H), 3.12-3.18 (m, 1H), 2.61 (ddd, J=52.5, 14.5, 6.5 Hz, 2H), 1.78-1.95 (m, 2H), 1.38-1.57 (m, 4H), 1.15-1.16 (m, 12H); ¹³C NMR (125 MHz, CDCl₃): δ 174.6, 172.4, 156.1, 143.9 (143.7), 141.2, 127.6, 126.99 (126.97), 125.12 (125.09), 119.9, 74.7, 66.9, 65.1, 53.6, 47.1, 45.2, 38.9, 31.9, 28.9, 28.2, 22.8, 22.3; IR (KBr): 3332, 3065, 2974, 2935, 2869, 1718, 1653, 1541, 1450, 1366, 1208, 1106, 1084, 1053, 989, 760, 740 cm⁻¹; HRMS (m/z): [M]⁺ calcd. for C₂₉H₃₉N₂O₆, 511.2808. found, 511.2787.

Fmoc-Lys(4-(tritylO) butyryl)-OH. Step 1: A mixture of 2.58 g (30 mM, 2.28 mL) of 4-butyrolactone and 1.2 g (30 mM) of sodium hydroxide in 30 mL of water was heated at 70° C. overnight. The clear solution was cooled and concentrated. The resulting white solid was suspended in toluene and concentrated further to remove the remaining trace amounts of water. An almost quantitative yield of sodium 4-hydroxybutyrate was obtained.

Step 2: Sodium 4-hydroxybutyrate (1.26 g, 10 mM) and trityl chloride (10 mM, 2.79 g) were dissolved in 30 mL pyridine for 3 days at 30° C. The solvent was evaporated and the residue was dissolved in ethyl ether. The ether solution was extracted with aqueous sodium hydroxide solution (4 g in 250 mL of H₂O). The aqueous solution was acidified to pH 3.0 and extracted twice with ethyl acetate. The combined organic phases were washed with brine and dried over anhydrous MgSO₄. The mixture was filtered and the filtration was evaporated to dryness give the solid product 4-(tritylO) butyric acid (1.29 g, 37%).

Step 3: DCC (3.7 mM, 760 mg) was added to a solution of 4-(tritylO) butyric acid (1.29 g, 3.7 mmol) and N-hydroxysuccinimide (3.7 mM, 425 mg) in 30 mL dioxane. The reaction was stirred at rt for 10 hrs. The solution was filtered and evaporated to dryness, and then the residue redissolved in 60 mL CH₂Cl₂. Et₃N (8 mM, 1.2 mL) and Fmoc-Lys-OH (4 mM, 1.62 g) were sequentially added. The mixture was stirred at rt for 4 hrs. After that, 150 mL water was added to the mixture and the solution was adjusted to pH 2.3. The organic layer was separated and the aqueous layer was extracted with EtOAc. The combined organic layers were dried over anhydrous Na₂SO₄. The solvent was evaporated and the residue was purified by flash column chromatography (MeOH/CH₂Cl₂=1/30). A yield of 1.5 g crude Fmoc-Lys(4-(tritylO) butyryl)-OH was obtained. The crude product contains more than 20% 1,3-dicyclohexylurea (DCU) based on the MS result, but it is pure enough for peptide synthesis. Fmoc-Lys(4-tritylO butyryl)-OH: ¹H NMR (500 MHz, CDCl₃): δ 8.03 (s, 1H), 7.69-7.72 (m, 2H), 7.49-7.57 (m, 2H), 7.31-7.42 (m, 7H), 7.23-7.29 (m, 12H), 5.88 (t, J=5.5 Hz, 1H), 5.83 (d, J=8.5 Hz, 1H), 4.28-4.44 (m, 3H), 4.09-4.20 (m, 1H), 3.04-3.17 (m, 4H), 2.28 (t, J=7.5 Hz, 2H), 1.67-1.94 (m, 4H), 1.26-1.39 (m, 4H); ¹³C NMR (125 MHz, CDCl₃): δ 174.8, 174.0, 156.2, 144.1 (144.0), 143.9 (143.7), 141.2, 128.6 (128.5), 127.8 (127.7), 127.6, 127.03 (127.01), 127.98 (127.93), 125.15 (125.09), 119.9, 86.6, 67.0, 62.5, 53.6, 47.1, 39.0, 33.7, 31.7, 28.9, 26.0, 22.1; IR (KBr): 3420, 3325, 3059, 2934, 2869, 1718, 1653, 1539, 1492, 1449, 1419, 1336, 1265, 1221, 1073, 760, 740, 707 cm⁻¹; HRMS (m/z): [M]⁺ calcd. for C₄₄H₄₅N₂O₆, 697.3278. found, 697.3268.

Results and Discussion

To search for possible novel histone marks, we analyzed a tryptic digest of core histones from HEK293 cells by HPLC/MS/MS. The generated MS/MS data were subjected to non-restrictive sequence alignment, searching for amino acid residues bearing mass shifts that were different from those of known PTMs. The analysis detected a previously undescribed mass shift of +86.0368 Da±0.02 Da (monoisotopic mass) at lysine residues of multiple histone peptides. This mass shift is therefore possibly caused by a new PTM. We used the accurately determined mass shift to predict the elemental composition of the modification (http://library.med.utah.edu/masspec/elcomp.htm), the most likely elemental composition for the modification moiety was C₄H₇O₂ (formula of mass shift plus one proton). This molecular formula has seven possible structural isomers: R- and S-isoforms of 3-hydroxybutyryl (3ohbu) (two possible enantiomers, the R- and S-isoforms, for the 3-hydroxybutyryl group), 3-hydroxyisobutyryl (3ohibu), R- and S-2-hydroxybutyryl (2ohbu), 2-hydroxyisobutyryl (2ohibu), and 4-hydroxybutyryl (4ohbu) (FIG. 1 a).

We used chemical methods to determine the structural isomers responsible for the detected mass shift. We first synthesized variants of two substrate histone peptides, K_(+86.0276)QLATK_(ac)AAR and PEPAK_(+86.0374)SAPAPK, incorporating each of the seven possible isomers at the sites of the mass shift. As expected, the synthetic K_(3ohbu)-containing H2BK5 peptides with the sequence PEPAK_(3ohbu)SAPAPK, either with a R-isoform, K_((R)-3ohbu), or S-isoform, K_((S)-3ohbu), co-eluted in HPLC/MS analysis, because enantiomers are impossible to be separated in a reverse-phase HPLC non-chiral column. Likewise, a mixture of K_((R)-3ohbu)QLATK_(ac)AAR and K_((S)-3ohbu)QLATK_(ac)AAR co-eluted, which was used for subsequent co-elution experiment. Because R-3-hydroxybutyryl-CoA is an important metabolite for lipid metabolism and R-3-hydroxybutyrate is a major component of ketone bodies, we chose lysine R-3-hydroxybutyrylation as the more likely candidate. In the rest of this paper, all the 3-hydroxybutyrylation and lysine 3-hydroxybutyrylation are referred as R-isoform instead of S-isoform unless specified.

The synthetic peptide, K_(3ohbu)QLATK_(ac)AAR, co-eluted with the corresponding in vivo-derived peptide, K_(+86.0276)QLATK_(ac)AAR, on HPLC, and had the same fragmentation pattern in HPLC/MS/MS (FIG. 2 a, b, and d). In contrast, the in vivo-derived peptide K_(+86.0276)QLATK_(ac)AAR had a different HPLC retention time than the other four synthetic structural isomers with identical mass, K_(3ohibu)QLATK_(ac)AAR, K_(2ohbu)QLATK_(ac)AAR (R/S mixture), K_(2ohibu)QLATK_(ac)AAR and K_(4ohbu)QLATK_(ac)AAR. Using the same method, we confirmed that the mass shift in the peptide PEPAK_(+86.0374)SAPAPK is also caused by lysine R-3-hydroxybutyrylation. Together, these data lead us to conclude that the identified mass shift of +86 Da is caused by lysine 3-hydroxybutyrylation, but not other structural isomers.

Next, we generated a pan antibody against R-3-hydroxybutyrylation (anti-K_(3ohbu)) using methods previously described, and used the antibody to further confirm lysine R-3-hydroxybutyrylation. The antibody showed good specificity in dot blot assay and competition experiments. In immunostained HEK293 cells, the PTM was mostly detected in nuclei. Treating the cells with sodium R-3-hydroxybutyrate at 10 mM, a concentration comparable to the range of 3-hydroxybutyrate concentrations in human diabetic ketoacidosis, dramatically enhanced nuclear lysine 3-hydroxybutyrylation. R-3-hydroxybutyrate and (R/S)-3-hydroxybutyrate (20 mM), but not S-3-hydroxybutyrate (20 mM), drastically induced Lys 3-hydroxybutyrylation in HEK293 cells, indicating that R-3-hydroxybutyrate is likely the main substrate leading to R-3-hydroxybutyrylation. Western blot analysis of whole cell lysate samples showed that K_(3ohbu) is present in Escherichia coli (strain ME9062), Drosophila melanogaster S2 cells, mouse embryonic fibroblast (MEF) cells and HEK293 cells (FIG. 3 a).

Because short-chain-CoAs are cofactors for a variety of lysine acylations, R-3-hydroxybutyryl-CoA may also be the cofactor for the lysine 3-hydroxybutyrylation reaction. R-3-hydroxybutyryl-CoA can be synthesized by several metabolic pathways (FIG. 1 b). Alternatively, it may be generated from cellular 3-hydroxybutyrate, possibly by 3-hydroxyacyl-Coenzyme A synthetase, in the same way that acetate and crotonate can be converted to their corresponding CoA derivatives. To test this hypothesis, we first treated HEK293 cells with 10 mM R-3-hydroxybutyrate and then examined lysine modifications by Western blot. We observed a dramatic increase in global Lys K_(3ohbu) in the treated cells compared to the control. Cells treated with crotonate also showed a slight increase in Lys K_(3ohbu), possibly caused by a slight increase in 3-hydroxybutyryl-CoA due to interconversion between crotonyl-CoA and 3-hydroxybutyryl-CoA. These results not only validate K_(3ohbu) but also imply that 3-hydroxybutyryl-CoA is the cofactor used for K_(3ohbu), just as acetyl-CoA and crotonyl-CoA are used for protein lysine acetylation and lysine crotonylation, respectively.

To confirm this possibility, we treated HEK293 cells with 20 mM isotopically labelled (R/S)-3-hydroxybutyrate [2,4-¹³C2], followed by HPLC/MS/MS analysis of histone peptides from the cells using a procedure similar to the one previously described. We found the isotopically labelled K_(3ohbu) peptides have an additional mass shift of 2 Da (e.g., 88.0328 Da vs 86.0276 Da in FIG. 2 c). In addition, these peptides have the same fragmentation patterns as the corresponding in vivo-derived and synthetic K_(3ohbu)-containing peptides (FIG. 2 a-c). Together, we identified 30 K_(3ohbu)-containing histone peptides bearing isotopically labelled K_(3ohbu) as validated by an additional mass shift of 2 Da.

3-hydroxybutyrate constitutes a major component of ketone bodies (FIG. 1 b) and its concentration can dramatically increase by more than 10-fold during starvation and over 20-fold in pathological conditions such as Type 1 diabetes (T1DM) and alcoholic liver damage (up to 20 mM). Thus, K_(3ohbu) levels may also change in response to an increased 3-hydroxybutyrate that may in turn enhance the concentration of 3-hydroxybutyrate CoA. To test this possibility, we examined K_(3ohbu) abundance by Western blot analysis using the liver samples from C57BL6 mice either fed with a normal chow or fasted (supplied with water only). Our results showed that K_(3ohbu) was drastically up-regulated after 48 hours of fasting (FIG. 3 b) relative to the control. The increase of K_(3ohbu) is congruent with the observed 7 fold increase in blood 3-hydroxybutyrate concentration in the fasted mice (to an average of about 3.5 mM, n=30) relative to the concentration in the non-fasted controls (which averaged about 0.5 mM, n=20) (FIG. 3 d). Consistent with this result, a similar increase in K_(3ohbu) status was observed in mice starved for 24 and 72 hours.

We further examined K_(3ohbu) in the streptozotocin (STZ)-induced type I diabetic mouse model. We observed a much more drastic increase of K_(3ohbu) in liver proteins (FIG. 3 c) in the STZ-treated mice compared to the control mice. We found that concentrations of blood glucose and 3-hydroxybutyrate in diabetic littermate mice increased by 2.4- and 10-fold, respectively, 48 hours after the mice were injected with STZ (200 mg/kg body weight; FIG. 3 e). Thus, in both fasted and STZ-treated mice, we observed a concerted increase of both 3-hydroxybutyrate concentrations and K_(3ohbu) levels.

Histone marks contribute to epigenetic mechanisms, playing a key role in diverse pathophysiological processes. To map major histone marks bearing K_(3ohbu), we analysed tryptic peptides of core histones from HEK293 cells treated with 10 mM R-3-hydroxybutyrate, and from liver cells of mice that were either fasted for 48 hours or treated with STZ. Together, we identified 45 histone K_(3ohbu) sites, including 38 histone K_(3ohbu) sites from 3-hydroxybutyrate-treated HEK293 cells (FIG. 4 a), 21 histone K_(3ohbu) sites from starved mouse liver (FIG. 4 a), and 16 histone K_(3ohbu) sites from STZ-treated diabetic mouse liver (FIG. 4 a). Among the 27 K_(3ohbu) sites identified in mouse liver cells, 11 of which were identified in both the STZ-treated diabetic mouse and starved mouse livers.

Identification of K_(3ohbu) substrates on a broad, cellular scale will reveal the scope of the modification and the pathways regulated by it. Additionally, development of a dataset of substrates will lay a foundation for studying non-chromatin functions of K_(3ohbu), as the history of the study of lysine acetylation demonstrates. To this end, we carried out a systematic analysis to identify non-histone substrates bearing K_(3ohbu) in HEK293 cells. Ten milligrams of whole-cell lysate was digested by trypsin and separated into 20 fractions with basic reversed phase HPLC. K_(3ohbu) peptides were affinity-enriched from each fraction using an anti-K_(3ohbu) antibody and then analysed by HPLC-MS/MS. We identified 3232 non-redundant K_(3ohbu) sites in HEK293 cells, with a false discovery rate of less than 1%. We then applied stringent cutoff criteria to further improve the quality of our identification. We removed all site identifications with a Maxquant peptide score below 60 and site localization probability below 0.9. The final dataset identified 3008 non-redundant K_(3ohbu) sites (3156 gene-based K_(3ohbu) sites) from 1359 proteins. The average mass error among these peptides was 0.069 ppm, with a standard deviation of 0.70 ppm.

Functional annotation analysis with gene ontology showed that the K_(3ohbu) proteome is significantly enriched in the nucleus (7.2E-192), intracellular lumen (2.5E-174), cytosolic ribosome (1.8E-22) and mitochondrial matrix (2.4E-12) (FIG. 4, b and c). K_(3ohbu) is abundant in proteins involved in diverse processes related to transcription and metabolism, such as nucleic acid metabolism (1E-130), gene expression (1.0E-97), macromolecular complex organization (5.0E-41), chromatin modification (1.2E-40) and DNA repair (1.6E-31) (table 6b). KEGG pathway enrichment analysis showed that the K_(3ohbu) proteome in HEK293 cells is significantly enriched in 16 complexes or pathways, including spliceosomes (7.2E-43), ribosomes (2.5E-19), RNA transport (2.0E-11), nucleotide excision repair (1.3E-8) and fatty acid elongation in mitochondria (2.3E-3). The spliceosome and ribosome stand out as heavily Lys 3-hydroxybutyrylated complexes when a protein-protein interaction map is constructed among K_(3ohbu) proteins. Analysis of the sequences surrounding K_(3ohbu) sites shows preferences for serine and proline at the −1 and +3 positions, respectively, and alanine and glycine at the +2 position (FIG. 4 d). This pattern is different from that seen for Lys acetylation.

To identify macromolecular complexes containing multiple K_(3ohbu) sites, we analysed complex enrichment using the CORUM database and identified over 70 complexes in which a significant proportion of subunits bear K_(3ohbu). Most of these complexes are in the transcription and RNA processing pathways, including the splicesome (3.5E-124), the ribosome (7.0E-59), the anti-HDAC2 complex (1.64E-27), the ALL-1 supercomplex (1.9E-24), the large Drosha complex (3.6E-23), the LSD1 complex (2.8E-17) and the MeCP1 complex (1.3E-17).

3-Hydroxybutyrate is generated mainly from oxidation of fatty acids in liver under physiological conditions such as starvation and during neonatal development when glucose is not sufficient¹. Ketosis can also happen when the insulin signaling pathway is not well regulated, as in Type 1 diabetes. During starvation, ketone bodies are important for generating acetyl-CoA as an alternative energy source for the brain and other tissues (e.g., heart and skeletal muscle). Given the dynamic nature of 3-hydroxybutyrate and of K_(3ohbu) levels, K_(3ohbu) may serve as a mechanism for cells to adapt to changes in cellular energy sources (e.g., glucose versus lipids) by rewriting epigenetic programs and modulating the functions of cellular proteins. Emerging evidence suggests that some KDACs have very weak deacetylation activities or activities other than deacetylation. It would be interesting to determine whether any of the KDACs can catalyse removal of 3-hydroxybutyrylation, therefore modulating cellular metabolism.

Several lines of evidence suggest that 3-hydroxybutyrate has functions other than simply providing energy. 3-Hydroxybutyrate has been used successfully to treat epilepsy. It also shows potential for treating several neurological conditions, such as Alzheimer's disease, Parkinson's disease, traumatic brain injury, ischemia, and amyotrophic lateral sclerosis. At the cellular level, R-3-hydroxybutyrate was found to modulate sperm motility, receptor signaling pathways, and autophagy, and to regulate global gene expression profiles associated with cancer cell “stemness”. Nevertheless, the molecular mechanisms by which R-3-hydroxybutyrate exerts these functions remain unclear. Discovery of the lysine 3-hydroxybutyrylation pathway therefore illuminates a new direction in studying the diverse physiological functions of R-3-hydroxybutyrate and its pharmacological significance.

The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate.

All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

TABLE 1 List of histone K3ohbu sites identified from HEK293 cells treated with 10 mM sodium R-3-hydroxybutyrate for 72 hours. “K(3ohbu)” and “K(ac)” indicates 3-hydroxybutyrylated lysine and acetylated lysine, respectively. Human SEQ histone ID protein Position Peptide Sequence NO H1 149 K(3ohbu)LAATPK 30 H1 23 VTK(3ohbu)AGGSAALSPSK 31 H1 34 AGGSAALSPSK(3ohbu)K 32 H1 143 GAPAAATAPAPTAHK(3ohbu)AK 33 H1 207 PSVPK(3ohbu)VPK 34 H1 85 LGLK(3ohbu)SLVSK 35 H1 90 SLVSK(3ohbu)GTLVQTK 36 H1 106 GTGASGSFK(3ohbu)LNK 37 H1 34 K(3ohbu)ASGPPVSELITK 38 H1 46 ASGPPVSELITK(3ohbu)AVAASK 39 H1 64 K(3ohbu)ALAAAGYDVEK 40 H1 97 GTLVQTK(3ohbu)GTGASGSFK 41 H1 75 ALAAAGYDVEK(3ohbu)NNSR 42 H1 168 K(3ohbu)PAAATVTK 43 H2A 5 AGGK(3ohbu)AGK(ac)DSGK 44 H2A 12 AGK(ac)DSGK(3ohbu)AK 45 H2A 120 K(3ohbu)TSATVGPK 46 H2A 128 TSATVGPK(3ohbu)APSGGK(ac)K 47 H2A 135 K(3ohbu)ATQASQEY 48 H2A 123 GK(3ohbu)LEAIITPPPAK 49 H2A 37 K(3ohbu)GNYAER 50 H2A 96 NDEELNK(3ohbu)LLGK 51 H2A 119 VTIAQGGVLPNIQAVLLPK 52 (3ohbu)K H2B 6 PEPTK(3ohbu)SAPAPK 53 H2B 12 SAPAPK(3ohbu)K(ac)GSK 54 H2B 17 GSK(ac)K(3ohbu)AVTK 55 H2B 21 AVTK(3ohbu)AQK 56 H2B 35 K(3ohbu)ESYSVYVYK 57 H2B 117 HAVSEGTK(3ohbu)AVTK 58 H2B 121 AVTK(3ohbu)YTSSK 59 H2B 6 PELAK(3ohbu)SAPAPK 60 H2B 17 K(3ohbu)AVTK(ac)VQK 61 H2B 24 AVTK(ac)VQK(3ohbu)K 62 H3 10 K(3ohbu)STGGK(ac)APR 63 H3 19, 24 K(3ohbu)QLATK(3ohbu)AAR 29 H3 19 K(3ohbu)QLATK(ac)AAR 29 H3 42 YQK(3ohbu)STELLVR 102 H3 24 QLATK(3ohbu)AAR 64 H3 28 K(3ohbu)SAPATGGVK 65 H3 57 YQK(3ohbu)STELLIR 66 H3 123 VTIMPK(3ohbu)DIQLAR 67 H3 28 K(3ohbu)SAPSTGGVK 68 H3 80 EIAQDFK(3ohbu)TDLR 69 H4 9 GGK(3ohbu)GLGK 70 H4 13 GLGK(3ohbu)GGAK(ac)R 71 H4 32 DNIQGITK(3ohbu)PAIR 72 H4 78 DAVTYTEHAK(3ohbu)R 73 H4 92 TVTAMDVVYALK(3ohbu)R 74

TABLE 2 List of histone K3ohbu sites identified from the livers of female C57BL/6 mice with 48 hrs starvation or C57BKS/J db/db mice treated with streptozotocin (200 mg/kg body weight) for 48 hrs. “K(3ohbu)” and “K(ac)” indicates 3-hydroxybutyrylated and acetylated lysine, respectively. Mouse SEQ histone ID protein Position Peptide Sequence NO H1 34 K(3ohbu)ASGPPVSELITK 38 H1 34 K(3ohbu)TSGPPVSELITK 75 H1 46 TSGPPVSELITK(3ohbu)AVAASK 76 H1 52 AVAASK(3ohbu)ER 77 H1 64 K(3ohbu)ALAAGGYDVEK 78 H1 64 K(3ohbu)ALAAAGYDVEK 40 H1 82 LVTTGVLK(3ohbu)QTK 79 H1 85 LGLK(3ohbu)SLVSK 35 H1 90 SLVSK(3ohbu)GTLVQTK 36 H1 97 GTLVQTK(3ohbu)GTGASGSFK 41 H1 106 GTGASGSFK(3ohbu)LNK 37 H1 164 VVK(3ohbu)VKPVK 80 H2A 12 AGGK(ac)AGK(ac)DSGK(3ohbu)AK 81 H2A 14 AGK(ac)DSGK(ac)AK(3ohbu)TK 82 H2A 96 NDEELNK(3ohbu)LLGK 51 H2A 96 NDEELNK(3ohbu)LLGR 83 H2A 116 ATIAGGGVIPHIHK(3ohbu)SLIGK 84 H2A 119 VTIAQGGVLPNIQAVLLPK(3ohbu)K 52 H2A 128 SSATVGPK(3ohbu)APAVGK 85 H2A 134 APAVGK(3ohbu)K 86 H2B 6 PEPTK(3ohbu)SAPAPK 53 H2B 6 PDPAK(3ohbu)SAPAPK 87 H2B 6 PELAK(3ohbu)SAPAPK 60 H2B 12 SAPAPK(ac)K(3ohbu)GSK(ac)K 88 H2B 12 SAPAPK(3ohbu)K(ac)GSK(ac)K(ac) 89 AISK H2B 12 PEPAK(ac)SAPAPK(3ohbu)K(ac)GSK 90 H2B 13 SAPAPK(ac)K(3ohbu)GSK(ac)K(ac) 91 AVTK(ac)AQK H2B 16 K(ac)GSK(3ohbu)K(ac)AISK 92 H2B 16 SAPAPK(ac)K(ac)GSK(3ohbu)K 88 H2B 16 K(ac)GSK(ac)K(3ohbu)AVTK(ac)AQK 93 H2B 17 K(ac)GSK(ac)K(3ohbu)AVTK 94 H2B 21 K(ac)GSK(ac)K(ac)ALTK(3ohbu)AQK 95 H2B 21 K(ac)AVTK(3ohbu)AQK(ac)K 96 H2B 24 AVTK(ac)VQK(3ohbu)K 62 H2B 24 K(ac)GSK(ac)K(ac)AISK(ac)AQK 97 (3ohbu)K H2B 35 K(3ohbu)ESYSVYVYK 57 H2B 109 LLLPGELAK(3ohbu)HAVSEGTK 98 H2B 117 HAVSEGTK(3ohbu)AVTK 58 H3 10 K(ac)STGGK(3ohbu)APR 63 H3 19 K(3ohbu)QLATK 99 H3 24 QLATK(3ohbu)AAR 64 H3 24 K(ac)QLATK(3ohbu)AAR 29 H3 80 EIAQDFK(3ohbu)TDLR 69 H3 57 YQK(3ohbu)STELLIR 66 H3 123 VTIM(ox)PK(3ohbu)DIQLAR 67 H4 6 GK(3ohbu)GGKGLGK(ac)GGAK(ac)R 100 H4 9 GGK(3ohbu)GLGK_ 70 H4 13 GLGK(3ohbu)GGAK 101 

What is claimed:
 1. An isolated peptide comprising a 3-hydroxybutyrylated lysine.
 2. The isolated peptide of claim 1, wherein the peptide is derived from a histone protein or a fragment thereof.
 3. The isolated peptide of claim 2, wherein the histone protein is derived from an organism selected from the group consisting of human, mouse, S. cerevisiae, Tetrahymena thermophila, D. melanogaster, and C. elegans.
 4. The isolated peptide of claim 1, wherein the peptide comprises an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-102.
 5. The isolated peptide of claim 1, wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 29-102.
 6. The isolated peptide of claim 1, wherein the peptide comprises at least 2 amino acid residues on each of the N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine.
 7. An isolated lysine 3-hydroxybutyrylation specific affinity reagent capable of binding specifically to the peptide of claim
 1. 8. The isolated lysine 3-hydroxybutyrylation specific affinity reagent of claim 7, wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 29-102.
 9. The isolated lysine 3-hydroxybutyrylation specific affinity reagent of claim 7, wherein the binding is dependent on the presence of the 3-hydroxybutyrylated lysine but not a surrounding peptide sequence thereof in the peptide.
 10. The isolated lysine 3-hydroxybutyrylation specific affinity reagent of claim 7, wherein the binding is dependent on the presence of the 3-hydroxybutyrylated lysine and a surrounding peptide sequence thereof in the peptide.
 11. The isolated lysine 3-hydroxybutyrylation specific affinity reagent of claim 7, wherein the lysine 3-hydroxybutyrylation specific affinity reagent is a protein.
 12. A method for producing the lysine 3-hydroxybutyrylation specific affinity reagent of claim 11, comprising screening a protein library using a peptide comprising a 3-hydroxybutyrylated lysine and at least two amino acid residues on each of the N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine.
 13. The isolated lysine 3-hydroxybutyrylation specific affinity reagent of claim 7, wherein the lysine 3-hydroxybutyrylation specific affinity reagent is an antibody.
 14. A method for producing the lysine 3-hydroxybutyrylation specific affinity reagent of claim 13, comprising immunizing a host with a peptide comprising a 3-hydroxybutyrylated lysine and at least two amino acid residues on each of the N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine.
 15. A method for detecting a 3-hydroxybutyrylated lysine in a protein or a fragment thereof, comprising: (a) contacting the protein or a fragment thereof with the isolated lysine 3-hydroxybutyrylation specific affinity reagent capable of binding specifically to the peptide of claim 1, whereby the lysine 3-hydroxybutyrylation specific affinity reagent and the protein or a fragment thereof forms a binding complex, and (b) detecting the binding complex, wherein the presence of the binding complex indicates the presence of a 3-hydroxybutyrylated lysine in the protein or a fragment thereof.
 16. The method of claim 15, wherein the lysine 3-hydroxybutyrylation specific affinity reagent is a protein.
 17. The method of claim 15, wherein the lysine 3-hydroxybutyrylation specific affinity reagent is an antibody.
 18. A method for determining the level of protein lysine 3-hydroxybutyrylation in a sample, comprising detecting a 3-hydroxybutyrylated lysine in the sample.
 19. A kit for detecting a 3-hydroxybutyrylated lysine in a protein of a fragment thereof, comprising the isolated lysine 3-hydroxybutyrylation specific affinity reagent capable of binding specifically to the peptide of claim
 1. 20. A kit for isolating a peptide containing a 3-hydroxybutyrylated lysine, comprising the isolated lysine 3-hydroxybutyrylation specific affinity reagent capable of binding specifically to the peptide of claim
 1. 